1. Field of the Invention
The present invention relates to a staining system for acid-fast bacilli. More particularly, the present invention relates to a quick stain for acid-fast bacilli which does not require a separate decolorization step utilizing an acid or alcohol.
2. Prior Art
The lipid containing cell walls of mycobacteria, such as M. tuberculosis, have the unique characteristic of binding carbolfuchsin stain so tightly that they resist destaining with strong decolorizing agents such as alcohol and strong acids. Thus, the term "acid-fast" has been used to describe a carbolfuchsin staining reaction for certain types of bacilli, such as mycobacteria which resists destaining by acids or alcohol. The acid-fast staining reaction of mycobacteria, along with their unique beaded and slightly curved shape, is a valuable aid in the early detection of infection and monitoring of therapy. The finding of acid-fast bacilli in the sputa or other mycobacteriological specimens is considered presumptive evidence of active tuberculosis and is sufficient to initiate therapy.
The present invention is an improvement in the types of acid-fast staining generally referred to as the Ziehl-Neelsen or "hot staining" method and the Kinyoun or "cold staining" method. For either method, a sample of sputum or other suitable mycobacteriological specimens, is used for preparing a culture. Preliminary digestion of the samples with pancreatin, typsin, dithiothreitol or other mucolytic substances increases the probability of isolating significant pathogenic bacteria. Since most of the bacteria found in acute pneumonia grow well on blood agar, this is the medium of choice. Inoculated plates are incubated for 18 to 24 hours and examined. Typical colonies are picked and stained with a gram stain for tentative identification of those organisms susceptible to gram staining.
In suspected cases of tuberculosis acid-fast staining techniques are used, since mycobacteria and other acid-fast organisms cannot be stained by gram stain. Smears are prepared in the same manner as for the gram stain on new slides. Slides are then treated by the Ziehl-Neelsen method or the Kinyoun method.
In the Ziehl-Neelsen method a carbolfuchsin stain is first prepared. The carbolfuchsin stain contains 0.3 grams of basic fuchsin, 10.0 milliliters of ethyl alcohol, and 90 milliliters of 5% aqueous solution of phenol. The carbolfuchsin stain is applied to the slide smears for 5 minutes, applying enough heat for gentle steaming. The stain is not permitted to evaporate and more stain is added as needed. The slides are then cooled and rinsed in water. The slides are then decolorized in a solution of 95% ethyl alcohol that contains 3% by volume of concentrated hydrocloric acid. The decolorizing solution is added until no more carbolfuchsin stain comes off. The slides are washed in tap water and are then counter-stained with a methylene blue solution for 1-2 minutes. The methylene blue solution contains 0.3 grams of methylene blue (90% dye content) and 100 ml of distilled water. The slides are then washed, dried and examined.
In the Kinyoun method the same counter-staining and methylene blue treatments are used as in the Ziehl-Neelsen method. The Kinyoun carbolfuchsin stain, however, contains 4 grams of basic fuchsin, 8 milliliters of liquefied phenol, 20 milliliters of ethyl alcohol (95%) and 100 milliliters of water. In the Kinyoun method the slide is fixed gently with heat and is then stained with the Kinyoun carbolfuchsin stain for 5 minutes without heating. The washing, decolorizing and counter-staining steps are the same as in the Ziehl-Neelsen method.
Numerous attempts have been made to minimize the time duration and number of steps involved in the Ziehl-Neelsen method and the Kinyoun method. In particular, it would be desirable to eliminate the acid decolorization step prior to counter-staining with methylene blue. None of the methods heretofore provided, however, have performed satisfactorily.
Accordingly, it is a primary object of the present invention to provide a quick acid-fast staining method which does not require a decolorizing step utilizing acid or alcohol. It is another object of the present invention to provide an acid-fast staining method which can be accomplished in less than about 10 minutes. It is another object of the present invention to provide a primary stain which can be utilized for acid-fast staining techniques which does not require heating.
These and other objects will become more apparent from the following description of the invention.